2012 Annual Report
1a.Objectives (from AD-416):
1. To define conditions to assure a 5 log reduction of acid tolerant pathogens in refrigerated or bulk stored acidified vegetables.
2. To determine how the metabolism of Escherichia coli O157:H7 (internal pH, membrane potential, ion concentrations, and cell metabolites) are affected as cells are exposed to organic acid and salt conditions typical of acidified foods.
3. To determine the survival of E. coli O157:H7 in commercial fermentation brines, with and without competing microflora, and under a variety of extrinsic and intrinsic conditions.
1b.Approach (from AD-416):
A cocktail of five or more pathogenic Escherichia coli O157:H7 strains from the USDA/ARS Food Science Research Unit culture collection will be used for these studies. While our previous work has focused on E. coli O157:H7 (from human, food, animal, and environmental sources) additional serotypes, including O145 strains obtained from ARS sources will also be used in this research. Previous research on acidified vegetable brines has shown that E. coli O157:H7 is the most acid resistant vegetative pathogen of concern for acidified vegetable products. E. coli O157:H7 and related serotypes can’t grow in most acidified vegetable products, the objective is to prevent bacterial pathogens from surviving long enough in non-heat treated acid and acidified foods to cause disease. Bacterial strains will be grown statically for 15 h at 37°C in non-selective broth (Luria broth) supplemented with 1 g/L glucose to induce acid resistance. Cell viability before, during and after acid treatments will be determined by plating on non-selective media to allow enumeration of injured cells with a spiral plater and an automated plate reader (Spiral Biotech). Samples from acid treatment of bacterial cells will be diluted in MOPS buffer at neutral pH prior to plating. The lower limit for detection is 10^2 to 10^3 CFU/mL for this method. In addition to standard plating, an MPN method done with microtiter plates, custom MatlabTM software, and a microtiter plate reader has been developed in our laboratory. This method can be used to determine log number for a range of cell concentrations from 10^8 to <30 CFU/mL, and will supplement spiral or standard plating techniques when cell numbers are lower than 10^3 CFU/mL. Most acid solutions will be prepared based on the protonated acid concentration. The acid concentration required to achieve specific protonated concentration for a given pH and ionic strength will be determined using a Matlab computer program (pHTools ) developed in our laboratory, or custom Matlab functions. Sodium gluconate will be used as a non-inhibitory buffer in acid solutions to allow comparisons of the effects of organic acids with the effect of pH alone. Cucumber juice medium or brined cucumbers will be used for these studies as representative of brined vegetable products, because these media do not contain inhibitors of microbial survival or growth, but do contain amino acids and other compounds that may aid in survival of the pathogens. Acid concentrations will be confirmed by HPLC using a Thermo Separation Products HPLC system with a Bio-Rad HPX-87H column and UV detector. For acid challenge experiments requiring anaerobic conditions, a Coy anaerobic chamber will be used and media or acid solutions allowed to equilibrate in the chamber for 24 h to remove dissolved oxygen.
Safe processing of acidified vegetables currently requires a 5-log reduction of bacterial food pathogens. Some acidified foods, such as refrigerate pickles, can’t meet the 5-log reduction standard with the current technology. Fumaric acid, which we have previously found to accelerate acid killing, was added to a refrigerated pickle formulation and found to effectively kill Escherichia coli O157:H7 within 9 days at 50°F and did not affect the acceptability of the flavor or texture. Fumaric acid was also used in semi-commercial scale trials with the natural preservative allyl isothiocyanate to preserve acidified cucumbers in a reduced salt brine. It was observed that fumaric acid is more effective than other traditional preservatives to prevent bacterial growth. The assessment of the use of lauric arginate to aid in the microbial stabilization of acidified cucumbers showed that this natural preservative is not robust enough to prevent the proliferation of microbes long term. Combinations of lauric arginate with natural preservatives for environmentally friendly preservation of cucumbers and for reduction in pathogen numbers in vegetable brines (without fermentation) are currently being evaluated. For fermented products we have previously shown that the killing of E. coli depends on pH. We, therefore, have investigated how chemical buffering changes during fermentation. A mathematical model for buffering was developed using Matlab software. The model was validated and the results from the model predicted the buffer components present in a complex acid solution. The model will have use in determining how pH changes during fermentation and competitive growth of bacteria. In addition to this work, preliminary studies to determine how cells of pathogenic E. coli strains are affected by organic acid solutions were done. A microbial metabolomics approach was devised to study the intracellular metabolite pools of E. coli O157:H7 which had been exposed to neutral pH, acid pH, or acid pH plus acetic acid. Cells exposed to acid pH utilize glutamate, supporting current theories for the acid resistance of E. coli O157:H7. Furthermore, 27 metabolites (including amino acids and nitrogenous metabolites) were found to decrease, and 20 to increase in response to acetic acid stress. The sources of experimental variation in the data were determined and the work provided a promising first look into the metabolism of acid-stressed E. coli O157:H7, which may be useful in developing enhanced preservation treatments for fruits and vegetables.
Development of a buffer capacity model to predict pH changes in acid and acidified foods. The growth of spoilage bacterial may increase pH in acidified foods, resulting in unsafe products. Conversely, pH reduction in fermented foods is important for assuring safety. However, there is currently no accurate method for predicting pH changes in acid and acidified foods because of the unknown buffering compounds which affect pH changes. To address this problem, ARS researchers at Raleigh, NC developed a buffer capacity model that can be used to determine pH changes in acid and acidified foods as acids and bases are added for processing, or are produced by the growth of bacteria. The model showed that the complex acid buffering present in commercial vegetable fermentation brines can be predicted even in the presence of unknown buffer compounds. Research to validate the model showed that pH changes in commercial fermented vegetables can be predicted. Spoilage related pH increase in acidified foods and beverages may be similarly modeled. By determining the buffer capacity of acid and acidified foods using the model, the potential health hazards that may occur (or be prevented) due to microbial activity could be predicted during production of acid and acidified foods, improving safety.